Demonstration of specific dust mite allergen induced response in a murine model

DEMONSTRATION OF SPECIFIC DUST MITE ALLERGEN-INDUCED RESPONSE IN A MURINE MODEL ONG SU YIN NATIONAL UNIVERSITY OF SINGAPORE 2007 DEMONSTRATION OF SPECIFIC DUST MITE ALLERGEN-INDUCED RESPONSE IN A MURINE MODEL ONG SU YIN (B. Sc. (Hons.) Biotechnology, UPM) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE, DEPARTMENT OF BIOLOGICAL SCIENCES, NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements Many important and wonderful people contributed towards making this body of work possible and this thesis will not have seen the light of day without their precious time, effort and assistance. With utmost gratitude, I would like to thank: • Assistant Professor Dr. Chew Fook Tim for his guidance, trust, understanding and patience in monitoring my work and progress throughout the entire duration undertaken. I am extremely grateful for his constant encouragement, crucial support and remain deeply humbled by the advice and lessons he generously imparted in the capacity as both supervisor, counselor and mentor; • Various personnel from the Laboratory Animal Centre of NUS, namely the staff of the Satellite Animal Holding Unit, Dr. Leslie Ratnam, and Dr. Enoka Bandularatne for their training and guidance regarding animal work; • Past and present fellow lab personnel from the Allergy and Molecular Immunology Laboratory of FGL, DBS, chiefly Dr. Ong Tan Ching, Ken Wong Kang Ning, Lim Puay Ann, Kelly Goh, and Kavita Reginald for their assistance and friendship; • Lastly but always, my beloved family: Atah and Mama, Su Ping and Su Gin, and dear respected peers: Yvonne Tan Yih Wan, Chan Siew Leong and Hema Jethanand, for their unconditional love, encouragement and belief in me. Their kinship, friendship and unwavering support provided the much-needed focus, motivation, and joyous moments to see through the challenging times. For them alone, I am truly blessed. i Table of Content Acknowledgements i Table of Content ii Summary iv List of Figures vi List of Tables vi List of Abbreviations vii 1.0 Introduction 1 1.1 Allergy and asthma 1 1.2 Dust mite allergens 4 1.3 Murine models of atopic asthma 8 1.4 Aims of this study 10 2.0 Materials and Methods 12 2.1 Production of recombinant allergens and allergen-specific 12 antibodies 2.1.1 Expression and purification of recombinant allergens 12 2.1.2 Generation of allergen-specific rabbit polyclonal antibodies 13 2.1.3 Generation of allergen-specific mouse monoclonal antibodies 13 2.2 Determining sera IgE reactivity of Singaporean atopic 14 population 2.2.1 Human serum samples 14 2.2.2 Immuno dot blot 15 ii 2.3 Quantification of dust mite allergens in Singaporean homes 16 2.3.1 Dust samples 16 2.3.2 Sample processing and allergen level quantification 16 2.4 Exposure of mice to recombinant allergens 17 2.4.1 Animals 17 2.4.2 Allergen exposure program 17 2.4.3 Measurement of airway hyperresponsiveness 18 2.4.4 Allergen-specific IgG1 and IgE quantification by ELISA 19 2.4.5 Lung histology 20 2.5 Approvals 21 3.0 Results and Discussions 22 3.1 IgE reactivity of Singapore atopic population 22 3.2 Distribution of allergens in environmental dust samples 25 3.3 Murine model of dust mite allergen-induced atopic asthma 29 3.3.1 Airway hyperresponsiveness (AHR) 30 3.3.2 Sera antibody profile response 38 3.3.3 Lung histology studies 41 4.0 Conclusion 43 References 48 iii Summary Dust mite allergens are important triggers of atopic asthma. Differential allergenic properties can however be observed in different groups of antigens. Existing lab research on recombinant allergens and available information on the IgE-binding capacity and distribution of dust samples in the environment of various dust mite allergen groups enabled us to postulate that these differences may be related to multiple factors including (exposure) level in the environment and inherent hostallergen interactions such as host airway and antibody responses to the allergens. In our assays, we used native Der p 1 and recombinant Der p 2, Blo t 3, Blo t 5, Der p 7, Blo t 12, and Der f 13 to represent each of the allergen group selected for the panel of study: Groups 1, 2, 3, 5, 7, 12, and 13. Atopic sera reactivity and house dust sample screens were carried out to profile the allergens in the local context. We then investigated the intrinsic nature of the dust mite proteins and the allergen-host interaction response by designing a murine model of atopic asthma. Host immune responses to each allergen were measured by airway hyperresponsiveness (AHR), sera allergen-specific antibody profile, and lung histology. For the IgE-binding capacity profiles, we conclude that allergens with both high capacity of IgE-binding were Der p 2, Der p 1 and Blo t 5 whereas allergens with low IgE-binding capacity are Der f 13 > Blo t 3 > Der p7 > Blo t 12 (magnitude) and Blo t 12 > Der p 7 > Blo t 3 > Der f 13 (frequency). From the environmental dust screens, Der p 1 and Der p 2 were categorized as having high environmental distribution levels, Blo t 5 and Der f 13 as moderate and Der p 7, iv Blo t 3 and Blo t 12 as poorly distributed. Groups 1 and 2 exhibited expected IgE and IgG1 production. However, Der p 1 did not induce any significant AHR trending compared to Der p 2. Comparisons between groups 2 and 13 can be drawn by their similarity in size and function. Interestingly, both groups demonstrated opposite effects on host AHR and antibody production. Blo t 12 also induced AHR suppression at high immunization doses, similar to Der f 13. Blo t 5 was able to induce increased AHR but only with immunization doses 5-fold higher. Der p 7 was able to induce increased AHR with elevated production of IgG1 at low immunization doses suggesting IgE tolerance. Groups 3 and 12 data corroborate them as minor allergens in comparison with major allergens such as groups 1 and 2. From the atopic population sera reactivity screens, the house dust distribution levels and the AHR responses were then analyzed to form a better profile of each allergen group. This study has demonstrated that each allergen group can exhibit differential host immunological responses and this may be attributed to the allergen’s intrinsic properties. v List of Figures Figure 1: Atopic asthma 3 Figure 2: The number of dust mite-sensitive individuals showing IgE reactivity to each recombinant allergen group. 23 Figure 3: IgE-binding of sera from Singaporean atopic individuals to 7 allergen groups. 24 Figure 4: Distribution of dust mite allergens in Singaporean homes. 26 Figure 5: Concentration of dust mite allergens in dust samples from Singaporean homes. 28 Figure 6: Der p 1-induced murine AHR. 31 Figure 7: Der p 2-induced murine AHR 32 Figure 8: Blo t 3-induced murine AHR 33 Figure 9: Blo t 5-induced murine AHR 34 Figure 10: Der p 7-induced murine AHR 35 Figure 11: Blo t 12-induced murine AHR 36 Figure 12: Der f 13-induced murine AHR 37 Figure 13: Allergen-induced murine sera IgG1 profile 40 Figure 14: Allergen-induced murine sera IgE profile 40 List of Tables Table 1: Dust mite allergens. 5 vi List of abbreviations Chemical and Reagents: BCIP 5-bromo-4-chloro-3-indolyl phosphate BSA bovine serum albumin IPTG isopropyl-β-thiogalactopyranoside NBT nitroblue tetrazolium TBS tris-buffered saline TMB 3,3,5,5-Tetramethylbenzidine Tris Tris (hydroxymethyl)-aminomenthane Units and Measurements hr hour(s) IU international unit kDa kilodalton M molar mg milligram min(s) minute(s) ng nanogram rpm revolution per minute U unit μg microgram μl microliter vii Others Ag antigen AHR airway hyperresponsiveness APC antigen-presenting cell EAACI European Allergy and Applied Clinical Immunology GST glutathione-S-transferase IgE immunoglobulin E IgG1 immunoglobulin G isotype 1 IL interleukin MW molecular weight PCR polymerase chain reaction pET expression vector (Novagen) RT room temperature spp. species Th T helper cell viii Chapter 1: Introduction 1.1 Allergy and asthma An antigen is any ubiquitous molecule that can be specifically recognized by the adaptive immune system and the specific recognition of it is the driving force of all adaptive immune responses. Often used interchangeably with the term antigen is the term allergen, which is defined as an antigenic substance capable of inducing an immediate type hypersensitivity reaction (i.e. allergy). Most people will have an immunological response to every antigen encountered in the environment but with varying degrees of response depending on his or her genetic predisposition that underlies the nature of response. Most responses are not harmful as the antigens are naturally cleared by the immune system but an atopic response may lead to clinical phenotypes such as allergic sensitization, clinical disease such as dermatitis, allergic rhinitis and chronic inflammatory responses such as asthma. There is currently no precise definition for atopy yet but a definition proposed by EAACI, (Johansson et al., 2001), stated that atopy is `a familial tendency to produce IgE antibodies to low doses of allergens, and to develop typical symptoms such as asthma, rhinoconjuctivitis, or eczema/dermatitis’. This atopic response or state of hypersensitivity induced by contact with a particular antigen (allergen) is commonly known as allergy and classified by Coombs and Gell (Coombs, 1975) as a type I hypersensitivity reaction. 1 Environmental allergens come from a variety of sources such as trees, grasses, fungi, food, mites, cats, dogs and bees. They are commonly found and widely distributed but a subset of only less than 1% to almost 10% of the population actually develops IgE responses to these allergens and go on to have a clinically significant allergic disease (Hayglass, 2003). However, this subset accounts for a pronounced cost on global health and quality of life. For example, an estimated 100–150 million people suffer from atopic asthma worldwide, and the disease claims 180,000 lives annually (Sly, 1999). The global expenditure for medical treatment of asthma is about USD12.7 billion per annum (Weiss and Sullivan, 2001). In Singapore, 1 in 5 school children (Goh et al., 1996) and 4% of the adult population (Ng et al., 1994) were reported to have asthma. More than 90% of patients with asthma and/or allergic rhinitis to dust mites and other inhalant allergens are found to be sensitized to Blomia tropicalis, Dermatophagoides pteronyssinus and Dermatophagoides farinae (Chew et al., 1999). Although asthma is a complex multifactorial disease, atopy presents a vital risk factor for asthma, especially with the most significant period of allergy sensitization development to allergens being in early childhood (Peden, 2002). A summary of the mechanism of allergy in the pathogenesis of atopic asthma is shown in Figure 1. 2 Figure 1: Atopic asthma (adapted from the HOPGENE Program for Genomic Applications; John Hopkins University USA, 2003 web resource) In order to induce allergy, sensitization must first take place. Atopic individuals usually already have existing specific antibodies circulating in their bloodstream, due to exposure to soluble allergens at mucosal surfaces from as young as early post-natal years (Niederberger et al., 2002; Kulig et al., 1999; Wahn et al., 1997). Upon uptake of allergen by antigen-presenting cells (APC), T cell–B cell interactions occur to induce specific B cells to switch immunoglobulin classes into IgE. IgE+ memory B cells and allergen-specific memory T cells are then established and boosted each time the allergen is repeatedly encountered. In an immediate phase reaction, cross-linking of effector cell-bound IgE by allergens releases biologically active mediators such as leukotrienes and histamines (e.g. mast cell degranulation), which causes symptoms of allergy. The late phase reaction occurs 2–24 hours after contact with allergen and involves proliferation of activated Th2 cells in response to the 3 allergens. Proinflammatory cytokines such as IL-4, IL-5 and IL-13 are released that promotes recruitment of eosinophils (Valenta, 2002). This early and late phase responses corresponds to what occurs in atopic asthma. 1.2 Dust mite allergens There are over 30 different proteins in a house dust mite extract that are able to induce IgE in dust mite-sensitive individuals (Thomas, 2002). To date, these proteins ranging from 7.2–114.0 kDa in molecular weight size have been classified into 21 groups (Table 1) based on their size, similarities in biochemical properties and sequence homology. The allergens are named according to the systematic nomenclature for disease-causing allergens that is formulated by a subcommittee of the World Health Organization (WHO) and the International Union of Immunological Societies (IUIS) and satisfy criteria of biological purity and allergenic importance (WHO/IUIS, 1994). Table 1 shows that among the described allergen groups of dust mites, group 1 and group 2 allergens which are known to be present in high concentrations in house dust (Custovic et al., 1996; Platts-Mills & Chapman, 1994), have the strongest IgE-binding capacity. Most of the dust mite allergen groups were identified in Dermatophagoides spp. followed by Blomia tropicalis and Lepidoglyphus destructor. 4 Allergen groupsa Biological Function MW (kDa) 1 Cysteine protease 25 IgE binding (%) 70–90 References 2 Unknown 14 60–90 Der f 2 (Trudinger et al., 1991), Der p 2 (Chua et a.,l 1990), Tyr p 2 (Eriksson et al., 1998), Eur m 2 (Smith et al., 1999), Gly d 2 (Gafvelin et al., 2001), Lep d 2 (Varela et al., 1994). 3 Trypsin 28,30 51–90 Der f 3 (Nishiyama et al., 1995), Der p 3 (Smith et al., 1994), Eur m 3 (Smith et al., 1999b)b, Blo t 3 (Cheong et al., 2003). 5 Unknown 15 9–70 Der p 5 (Tovey et al., 1989), Blo t 5 (Arruda et al., 1995), Lep d 5 (Eriksson et al., 2001). 6 Chymotrypsin 25 30–40 Der f 6 (Kawamoto et al.,1999), Der p 6 (Yasueda et al., 1993). 7 Unknown 22–31 50–62 Der p 7 (Shen et al., 1993), Der f 7 (Shen et al., 1995), Lep d 7 (Eriksson et al., 2001). 8 Glutathione-Stransferase 26 40 Der p 8 (O'Neill et al., 1994). 9 30 >90 Der p 9 (King et al.,1996). 10 Collagenolytic serine protease Tropomyosin 33–37 5-80 Der p 10 (Asturias et al., 1998), Der f 10 (Aki et al., 1995), Blo t 10 (Yi et al., 2002), Lep d 10 (Saarne et al., 2003). 11 Paramyosin 92,98, 110 80 Der f 11 (Tsai et al., 1999), Der p11 (Tategaki et al., 2000), Blo t 11 (Ramos et al., 2001). 12 Unknown 14 50 Blo t 12 (Peurta et al., 1996). Der f 1 (Dilworth et al., 1991), Der p 1 (Chua et al., 1988), Der m 1 (Lind et al., 1988), Eur m 1 (Kent et al., 1992), Blo t 1 (Mora et al., 2003). 5 13 Fatty acid binding protein 14,15 10-23 Blo t 13 (Caraballo et al., 1997), Lep d 13 (Eriksson et al., 2001), Aca s 13 (Eriksson et al., 1999). Der f 13 (Chan et al., 2006) 14 Apolipophorin 177 30 c 39 d 70 e Der f 14 (Fujikawa et al., 1996), Eur m 14 (Epton et al., 1999), Der p 14 (Epton et al., 2001). 15 98 kDa Chitinase Gelsolin-like protein/villin 98 ? Der f 15 (McCall et al., 2001). 53 35 Der f 16 (Tategaki et al., 2000). 17 EF-hand calciumbinding protein 53 35 Der f 17 (Tategaki et al., 2000) 18 60kDa chitinase 60 54 Der f 18 (Weber et al., 2003) 19 Anti-microbial peptide homologue. 7,2 ? Blo t 19b 20 Arginine kinase Unknown 40 ? Der p 20b 15 ? Der p 21b Heat shock protein 70 kDa 70 10 Der f (Aki et al., 1994) 16 21 Mag 29f Table 1: Dust mite allergens. Species name of dust mites: Der f (D. farinae), Der p (D. pteronyssinus), Der m (D. microceras), Eur m (Euroglyphus maynei), Tyr p (Tyrophagus putrescentiae), Lep d (Lepidoglyphus destructor), Gly d (Glycyphagus domesticus), Blo t (Blomia tropicalis), and Aca s (Acarus siro). a Listed in the WHO/IUIS list of allergens as of June 2006 (http://www.allergen.org/List.htm) b Unpublished but sequence data available in WHO/IUIS list of allergens or GenBank. c Data for Mag allergen d Data for recombinant Mag 3 allergen e Data for natural Mag 3 allergen f Not listed in WHO/IUIS list of allergens but published and sequence data available in GenBank. 6 More than 95 % of the allergen accumulated in mite cultures is found in fecal particles (Tovey et al., 1981), which makes mite feces a major source of house dust allergen. Dust mite allergens have already been detected in household niches worldwide. For an atopic individual, it takes lesser amounts of allergens to invoke an immune response compared to a non-atopic individual. Studies have previously been conducted and are also ongoing to correlate the amount of allergen found in environmental dust with the risk of allergen sensitization. Many functions of the dust mite allergen groups have been elucidated except for groups 2, 5, 7, 12 and 21. Their diverse biological functions include enzymes, enzyme inhibitors, ligand binding proteins and structural proteins. Dust mite allergens are one of the most important aeroallergens inducing asthma and are much more relevant than ovalbumin which is the standard antigen used in murine models of atopic asthma. There is also a lack of animal models using dust mite allergens as the allergen source (Sharma et al., 2003). The available studies of atopic asthma using dust mite allergens have mostly been limited to house dust mite extracts (Tategaki et al., 2002; Tumas et al., 2001) rather than the use of recombinant proteins. The content of extracts includes a variety of allergenic and non-allergenic components which are often difficult to standardize or ensured free of contamination from other non-dust mite proteins. Positive reactions to a given allergen extract will indicate that an allergic subject is sensitized against extract components without identifying the specific components. Hence, the use of recombinant allergens allows for specific quantification of host response to allergen groups investigated. 7 1.3 Murine models of atopic asthma The Mouse Genome Project has revealed that mice and humans both have about 30,000 genes and share 99% of those genes alike. About 1,200 new genes were discovered in the human genome because of mouse-human comparisons (90 % of genes associated with diseases are identical in human and mouse). The availability of well-characterized mutants and inbred strains provide a wealth of information and opportunities (Renz et al., 2002). Different strains vary in phenotypes and susceptibility to disease induction, echoing the heterogeneity in humans (Gosselin et al., 2002). There are also many available antibodies and reagents that are specific to the mouse. These collectively make the mouse a very useful model to study the pathogenesis of human diseases. In the last decade itself, many advances in understanding the mechanism of asthma and allergy have been made with the use of murine models. These studies have also proven useful in characterizing specific allergen-induced immunological responses and immunological properties of allergens. BALB/c and C57BL/6j are two of the commonest strains of mice used in studies of allergies and atopic asthma. One of the main factors to consider when choosing a strain is its airway responsiveness to allergen-induced challenges. The rank of order for airway responsiveness among inbred murine strains is already well studied: A/J > BALB/c > C3H/HeJ > C57BL/6j (Duguet et al., 2002). Sensitized BALB/c mice have greater AHR compared to C57BL/6j mice (Brewer et al., 1999; Zhang et al., 1999). BALB/c mice develop allergen-induced Th2-cytokines gene expression, airway inflammation and hyperresponsiveness 8 whereas C57BL/6j mice are less reactive (Gosselin et al., 2002; Yip et al., 1999) making them suitable for comparison work between a responder and non-responder strain. Different laboratories perform murine experiments differently in studying atopic asthma as there is no standard experimental protocol which is also not feasible considering the vast kinds of studies that are performed with different variables. Each experimental protocol is usually designed to exhibit the hallmark features of a murine model of atopic asthma which are bronchial eosinophilic inflammation and airway hyperresponsiveness (AHR) (Leong & Hudson., 2001). To produce a murine model, mice are usually injected with an antigen to induce systemic sensitization before the same antigen is then administered through the airways to focus the inflammatory process in the bronchi and lungs. Mice exposed to an antigen only through the respiratory route develop AHR without histologic airway inflammation (Hessel et al., 1995 & Renz H, et al. 1992). Systemic antigen sensitization with the use of an adjuvant or intratracheal challenge is the most common antigen administration route used (Tumas et al., 2001). Allergen-specific IgE is measured as risk factor for asthma as well as the allergenicity of the allergen whereas IgG1 is measured because it is also able to bind mast cells and basophils to cause degranulation (Tumas et al., 1991). Experimental protocols mostly differ in the age of animals used, dose and type of allergen, route of allergen administration, length of allergen exposure and method of measuring AHR. These differences tend to make comparison of published results difficult. For example, the dose of ovalbumin (a commonly investigated allergen) for systemic sensitization varies from 1 μg (Nakajima et al., 9 1994) to 8,000 μg (Wilder et al., 1999). Furthermore, in a dose-comparison study, 10 μg of ovalbumin established a working asthma model, but 1,000 μg failed to do so (Sakai et al., 1999). Commonly measured parameters include sera allergenspecific immunoglobulin levels, AHR, lung histology, cell infiltration counts, and bronchoalveolar lavage fluid (BALF) cytokine levels. 1.4 Aims of this study This thesis focuses on characterizing the immunological properties of 7 selected dust mite allergen groups in the context of elucidating the pathogenesis of atopic asthma. The dust mite allergen groups 1, 2, 3, 5, 7, 12 and 13 were selected based on published IgE-binding profiles for each allergen and house dust distribution data as well as their biological functions (known or putative). Some groups are similar and yet most are different. Together they represent the dust mite allergens across a broad spectrum of immunogenicity. In our assays, the allergen groups mentioned were represented by native Der p 1 and recombinant Der p 2, Blo t 3, Blo t 5, Der p 7, Blo t 12 and Der f 13. We were interested to find out if the specific immunogenicity of each allergen group depended on its IgE-binding capacity, its concentrations in the environment and/or its intrinsic biochemical properties. Therefore, we investigated the sera reactivity of a selected Singaporean atopic population to these allergens and the respective allergen distribution and concentrations in local Singaporean households. We were also intrigued by how each of the selected allergen groups would interact with the immune system of an atopic host. What 10 are the differences or similarities between the groups in the context of inducing an immunological response? Therefore, we have chosen a murine model of atopic asthma to characterize host immunological responses to the allergen groups such as serum antibody and airway responses. A primary motivation for this study is that dust mite allergens present the highest sensitization risk for atopic and childhood asthma in our local population of Singapore. Through this study, we aim to better understand the mechanisms of allergy sensitization and the role of dust mite allergens in the pathogenesis of atopic asthma. This study also provided materials (lungs, sera and BAL fluid from mice immunized with and exposed to dust mite allergens) for future functional genomic and proteomic characterization of dust mite allergen-induced responses in a host immune system. Such future characterization will yield possible clues into putative molecular markers or pathways of target in the pathogenesis of atopic asthma. The main deliverables of this study were local population sera IgE-binding reactivity profiles of the allergens, the concentrations of the allergen groups in the local environment and the specific immunological responses elicited by these allergen groups as measured by airway hyperresponsiveness (AHR), serum antibody profile and lung histology. 11 Chapter 2: Material and Methods 2.1 Production of recombinant allergens and allergen-specific antibodies 2.1.1 Expression and purification of recombinant allergens Protein expression of soluble recombinant allergens was carried out by transforming plasmids containing DNA inserts of wild type allergens into E. coli strain BL21 (DE3) cells. 1.0 mM IPTG was used to induce the cultures at 37 ˚C for 4 hrs with constant shaking at 200 rpm. The induced cultures were centrifuged to collect the bacterial cells (5000 rpm, 20 mins, 4 ˚C), then resuspended in binding buffer (5mM imidazole, 0.5M NaCl, & 20mM Tris-HCl pH 7.9). Cells were then lysed by sonication to obtain the recombinant proteins. The supernatant from the pelleted lysate was purified using Ni-NTA resin (Novagen; USA) under denaturing conditions and eluted from the Ni-NTA resin using elution buffer (1M imidazole, 0.5M NaCl, & 20mM Tris HCl pH 7.9). Bacterial cells containing insoluble recombinant allergens were resuspended in binding buffer (5mM imidazole, 0.5M NaCl, & 20mM Tris-HCl pH 7.9) added with 6 M guanidine hydrochloride. The proteins were then refolded by rapid dilution into their respective buffers or PBS at 4ºC. The refolded proteins were concentrated using Amicon® Stir Cell (Millipore; USA) using membranes (Millipore; USA) with suitable molecular weight cut-off pores. Purified recombinant proteins were stored at 4 ºC for immediate use and at -80 ˚C for long term storage. Protein concentration was measured using the Bradford assay, with BSA as the standard. 12 2.1.2 Generation of allergen- specific rabbit polyclonal antibodies New Zealand White Rabbits (2.5 to 3 kg) were purchased from the Centre for Animal Resources, Singapore and housed in the university Animal Holding Unit. Food and water were provided ad libitum. Animals were sacrificed by chemical euthanasia after the final harvest and disposed off as biohazard waste according to biosafety guidelines. Immunization was administered to the animals subcutaneously using 300 μg of recombinant protein diluted in a mixture of 500 μl of PBS and equal volume of Freund’s complete adjuvant (Sigma-Aldrich; Germany). Booster shots were repeated every 3 weeks with the same amount of recombinant protein, but using incomplete Freund’s adjuvant (Sigma-Aldrich; Germany) instead. All animals were housed at the Animal Holding Unit in the National University of Singapore throughout the duration of the antibody production work. After each booster shot, blood samples were obtained and animal antibody levels are titered using ELISA. A final harvest of blood was collected once the antibody titer was sufficiently maintained and the animals were finally sacrificed. The harvested blood was allowed to clot overnight at 4oC. Subsequently it is centrifuged at 3000 x g for 20 mins to obtain the sera, which were then stored in at -20oC. 2.1.3 Generation of allergen- specific mouse monoclonal antibodies 8 weeks-old female SPF BALB/c mice were purchased from the Centre for Animal Resources, Singapore and housed in the university Satellite Animal 13 Holding Unit. Food and water were provided ad libitum. Animals were sacrificed by carbon dioxide overdose after the final harvest and disposed off as biohazard waste according to biosafety guidelines. Mice were immunized intraperitoneally with 25 µg of each allergen (as described in 2.1.1) in Immuneasy Mouse Adjuvant (Qiagen; Germany) and boosted every three weeks until high titers of allergenspecific antibodies were obtained. The hybridomas were produced by polyethelene glycol (PEG) fusion of myeloma cells and splenocytes from immunized mice in a ratio of 3:1. Hybridoma clones were screened through HAT (hypoxanthine, aminopterin and thymidine) (Sigma; Germany) selection followed by HT (hypoxanthine and thymidine) (Sigma; Germany) medium. The hybridoma clones were then screened with both whole mite extract and the specific allergens using enzymatic immuno assays. Producers were cloned twice by limiting dilution and the selected clones were further expanded in vitro. 2.2 Determining sera IgE reactivity of Singaporean atopic population 2.2.1 Human sera samples In this study, a collection of consecutive serum samples over a one year period from Singaporean patients with atopic clinical profiles were used for IgE reactivity screening. The sera were also screened for dust mite allergen reactivity by assaying with crude protein extracts of D. pteronyssinus, D. farinae and B. tropicalis. 14 2.2.2 Immuno dot blot For each serum sample, 1 μg of each recombinant protein to be assayed were dotted on a nitrocellulose membrane (BIO-RAD Laboratories; USA). The membrane was allowed to dry at RT before being blocked with PBS-0.1% Tween20 for an hr. Following this, the membranes were incubated in dust mite reactive atopic patients’ sera overnight at 4ºC, followed by goat anti-human IgE conjugated with alkaline phosphatase (Sigma-Aldrich; Germany) diluted 1:1000 for 2 hrs. Colorimetric reactions on the membranes were then detected by incubating with NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate) (Promega; Madison, CA, USA). The Olympus MicromageTM for Windows version 3.01 (Olympus Optical; Germany) image analysis software was used to quantify the dot intensities. All membranes were washed three times with wash buffer (PBS-0.05 % Tween-20) in between every step of this assay before colour change detection. IgE-binding reactivity was categorized based on optical density (OD) of the immuno dot blot reactions: high (OD > 100), medium (50 > OD < 100), low (20 < OD < 50) and negative (OD < 20) from the maximum score of 255. 15 2.3 Quantification of dust mite allergens in Singaporean homes 2.3.1 Dust samples Dust samples were collected from mattress, kitchen, sofa, carpet and bedroom floor areas of volunteer homes in Singapore. This collection was a separate exercise and did not correlate with the homes of atopic patients studied (refer to section 2.2.1.). Volunteer homes were randomly selected from around Singapore. An area of 1m2 for each area sampled was vacuumed for 2 mins using a modified Kirby Classic III vacuum cleaner (Kirby Co.; USA). 2.3.2 Sample processing and allergen level quantification Dust samples collected were firstly sieved using a 500 μm pore-sized sieve. For every 50 mg of dust sample, 1 ml of PBS was added and then the solution was incubated overnight at 4ºC with shaking. The samples were then centrifuged at 2500 rpm for 20 mins at 4ºC, and the supernatant collected was stored at -20 ºC. The supernatant (100 μl )of each individual dust samples was coated overnight at 4°C onto monoclonal antibody-coated (as described in 2.1.3) wells in a microtiter plate (NUNC; Denmark) after the plate had been blocked with 1 % BSA in PBS for 30 mins at RT. Throughout the assay, wells were washed thrice with PBS-T (0.05 %) in between steps. Subsequently the wells were incubated overnight at 4°C with 100 µl of anti-allergen rabbit IgG antibodies (as described in 2.1.2) at 1:5000 dilutions in PBS. Wells were then washed and incubated with 1:1000 16 dilution of anti-rabbit IgG-conjugated horseradish peroxidase (BD Pharmingen; USA) in PBS for 3 hrs at RT. Wells were rinsed completely before addition of TMB (Sigma; USA). Finally, reactions were stopped using 20 μl of 1 M HCl and plates were read at 450 nm. 2.4 Exposure of mice to recombinant allergens 2.4.1 Animals 7 week-old female SPF BALB/c mice were purchased from the Centre for Animal Resources, Singapore and housed in the university Satellite Animal Holding Unit. Mice were housed in separate cages according to treatment. Food and water were provided ad libitum. Animals were sacrificed by cervical dislocation under anesthesia at the completion of the study and disposed off as biohazard waste according to biosafety guidelines. 2.4.2 Allergen exposure program Mice were held for a week before the commencement of the allergen exposure program. At the end of the holding period on day 0, mice were bled via the retro orbital sinus to obtain pre-exposure sera and screened for airway hyperresponsiveness (as described in 2.4.3; native Der p 1 from Indoor Biotechnologies; UK). Mice were sensitized on days 1 and 15 with an intraperitoneal injection of 0.5, 1, 2 or 4 μg of recombinant allergen protein (as 17 described in 2.1.1) suspended in PBS to a total volume of 200 μl. Subsequently, mice were administered daily challenges from day 16 through to day 19 using intranasal application of 1 μg of the same recombinant allergen protein suspended in PBS to a total volume of 50 μl. Non-sensitized animals received only the PBS solution. A final bleed was performed on day 20 to obtain post-exposure sera. All bleeding and intranasal challenges were performed under anesthesia. Harvested whole blood was allowed to clot overnight at 4oC before being centrifuged at 3000x g for 20 mins to obtain the sera. 2.4.3 Measurement of airway hyperresponsiveness Single-chamber whole body plethysmographs (Buxco Electronics, Inc.; Wilmington, NC, USA) were used to measure pulmonary function without the use of anesthesia or restraint on the animals. Airway resistance is expressed as Penh units using this non-invasive method. Mice were challenged on day 20, 24 hrs post-allergen challenge, with increasing doses of aerosolized methacholine (2.5, 5, 10, and 20 mg/ml) and pulmonary functions were recorded. The initial Penh reading when animals were exposed to only 400 μl of aerosolized PBS solution for 2 mins was recorded as baseline Penh. An aerosol challenge in increasing methacholine dose (400 μl for each concentration) was administered via the Buxco Aerosol Delivery Unit (Buxco Electronics, Inc.; Wilmington, NC, USA) with duty cycle of 33 % for exactly 2.5 mins followed by a 0.6 min drying period for each dose. Animal pulmonary response data were then recorded for 5 mins and a mean of this period in terms of Penh was obtained. All Penh values for each 18 mouse were allowed to return to baseline before the next higher dose of methacholine was administered. The results of methacholine challenges were expressed as the percentage above baseline Penh index. 2.4.4 Allergen-specific IgG1 and IgE quantification by ELISA For the quantification of allergen-specific IgG1, allergen proteins (as described in 2.1.1) were coated overnight at 4˚C onto Maxisorp ELISA plate (NUNC; Denmark) at 0.5 μg per well (50 μl) in carbonate buffer. Plates were washed with PBS-T (PBS, 0.05 % Tween-20) and blocked with 100 μl of PBS-0.1 % Tween20-0.01 % BSA for 2 hrs at 37ºC. Murine sera samples were applied in dilutions of 1:100, 50 μl per well and in duplicates before being incubated at 4ºC overnight. HRP-conjugated anti-IgG1 (Zymed Laboratories Inc.; USA) were then applied, 50 μl per well in 1:2000 dilution and incubated at RT for 2 hrs. Microtitre plates were thoroughly washed thrice with PBS-T between each step. Colorimetric reaction was developed for approximately 30 mins with the addition of 100 μl of TMB substrate (Sigma-Aldrich; Germany). Finally, the reaction was stopped by adding 20 μl of 1 M HCl per well. Absorbance was measured at 450 nm using an ELISA plate reader. For the quantification of allergen-specific IgE, a 5-layer sandwich ELISA method was used due to the scarce IgE titers. Anti-mouse IgE monoclonal antibodies (BD Pharmingen; USA) were coated overnight at 4˚C onto Maxisorp ELISA plates (NUNC; Denmark) at 100 ng per well (50 μl) in carbonate buffer. Plates were washed with PBS-T (PBS, 0.05 % Tween-20) and blocked with 100 μl of PBS-0.1 % Tween-20-0.01 % BSA for 2 hrs at 37ºC. This is followed by 19 overnight incubation with mouse sera at ½ dilutions and a subsequent overnight incubation with specific allergens at 125 ng per well (2.5 μg/ml). The final overnight incubation with allergen-specific IgG (as described in 2.1.2) using either 1:500 or 1:1000 dilutions was performed before addition of HRP-conjugated antirabbit IgG (BD Pharmingen; USA) at 1:2000 dilutions and incubated at RT for 2 hrs. Detection method was as described earlier for the quantification of IgG1 and plates were washed thrice with PBS-T between each step throughout the assay. 2.4.5 Lung histology Upon completion of the methacholine challenges (as described in 2.4.3), mice were sacrificed by cervical dislocation under anesthesia. Mouse lungs were washed with 1 ml of PBS and distended with 10 % formalin solution. The collected tissues were then processed for microscopy. First, the lungs were dehydrated with a series of alcohol followed by clearing of dehydrant with histoclear. Then the tissues were infiltrated with paraffin wax as the embedding agent. Tissues embedded in wax were then sectioned using a microtome into samples of 5–7 microns thickness and mounted on microscopic slides. Dewaxing was then done to allow penetration of water-soluble dyes. The prepared murine lung sections were stained with hematoxylin and eosin dyes. Slides were analyzed under low power (X 10) for determination of lung tissue inflammation and for eosinophilic infiltration at high power magnification (X 40). 20 2.5 Approvals All protocols involving human sera were reviewed and approved by the Institutional Review Board of the Singapore General Hospital and the Hospital Ethics Committee of the KK Women's and Children's Hospital. Prior consent was obtained from owners of volunteer homes involved in the environmental dust sampling study. All animals used and animal research protocols were approved by the International Animal Care and Use Committee (IACUC) and the Animal Research Ethics Committee of the National University of Singapore. 21 Chapter 3: Results and Discussion 3.1 IgE reactivity of Singapore atopic population Sera from 162 atopic individuals in Singapore were assayed for IgE reactivity to the study panel of 7 allergen groups, comprising allergens: Der p 1, Der p 2, Blo t 3, Blo t 5, Der p 7, Blo t 12 and Der f 13. Specific serum IgE reactivity to each allergen was measured using colorimetric-based immuno dot blot assays. The sera reactions were quantified by unit optical density (OD), the unit of absorbance of which is directly proportional to the percentage of IgE binding. IgE-binding levels were then categorized as negative (OD < 20), low (20 < OD < 50), moderate (50 < OD < 100), and high (OD > 100). 114 (69.1%) out of the 162 atopic sera samples were determined as dust mite-sensitive sera by assaying with crude proteins of D. pteronyssinus, D. farinae and B. tropicalis. 50.9% of the dust mite-sensitive atopic individuals had positive reactions to Der p 2, followed by 39.5 % to Der p 1 and 35.1 % to Blo t 5. For the other allergens, reactions towards Blo t 3, Der f 13, Der p 7, and Blo t 12 were 15.8 %, 19.3 %, 21.05 % and 26.3 % respectively (Figure 2). High positive reactions towards Der p 1 and Der p 2 were expected due to the known identification of group 1 and 2 allergens as major allergens with high IgE-binding frequencies (Chapman et al., 1980; Van der Zee et al., 1988; Lind et al., 1983). Serum IgE reactivity for Blo t 5 which is identified as a major allergen of B. tropicalis (Caraballo et al., 1996) ranked third highest among the allergens screened. These data correlated with those among the atopic population 22 of tropical Singapore, Dermatophagoides (Lee et al., 1994 & 1989) and Blomia (Lee et al., 1997 & 1996) allergens are co-sensitizers (Fernandez-Caldas et al., 1998; Hage- Hamsten et al., 1995). 19.3% of the dust mite-sensitive atopic sera responded towards Der f 13, despite D. farinae representing only about 1 % of mite fauna found in Singapore (Chew et al., 1999). This is most probably due to the cross-reactivity of Der f 13 with other group 13 allergens. 100% 90% 80% 70% Neg 20 50% Med >50 40% High >100 30% 20% 10% 0% Der p Der p Blo t 3 Blo t 5 Der p Blo t 1 2 7 12 Der f 13 Figure 2 The number of dust mite-sensitive individuals showing IgE reactivity to each recombinant allergen group. Reactive sera were defined as sera with positive OD of > 20. The percentage of patients reacting to each allergen is calculated based on 114 dust mite-sensitive individuals from a total of 162 Singaporean atopic individuals screened. 23 The bulk of the positive sera reactions for each allergen was of the low reactivity category (20 < OD < 50) (Figure 3). Der p 2 had the highest number of reactors for high, moderate and low categories. Among the panel of allergens investigated in this study, we can conclude that allergens with both high frequency and magnitude of IgE-binding are Der p 2, Der p 1 and Blo t 5. Among the allergens with low IgE-binding capacity were Der f 13 > Blo t 3 > Der p7 > Blo t 12 (magnitude) and Blo t 12 > Der p 7 > Der f 13 > Blo t 3 (frequency). Optical Density (OD) 200 high 150 100 moderate 50 low 0 negative Der p 1 Der p 2 Blo t 3 Blo t 5 Der p 7 Blo t 12 Der f 13 Allergen Figure 3 IgE-binding of sera from Singaporean atopic individuals to 7 allergen groups. The percentage of IgE-binding is expressed as optical density (OD) and categorized by specific reaction levels: low (20 < OD < 50), moderate (50 < OD < 100) and high (OD > 100). Sera with reaction OD < 20 were considered negative. 24 Atopic serum IgE reactivity profiles of dust mite-sensitive individuals tells us which dust mite allergens in our panel of study are Singaporeans reacting to and the amount of sensitization. Identifying specific dust mite allergens will aid in the study of the role of host response in disease pathogenesis. We then proceeded to investigate if the degree of dust mite sensitization is due to concentration levels in the local environmental dust, to the intrinsic allergenic property of the respective allergen proteins or a combination of both. 3.2 Distribution of allergens in environmental dust samples In order to investigate if the degree of dust mite sensitization is due to exposure levels in the local environment, dust samples were collected from Singaporean homes and assayed with ELISA for concentrations of Der p 1, Der p 2, Blo t 3, Blo t 5, Der p 7, Blo t 12 and Der f 13, representing the major dust mite allergen groups of 1, 2, 3, 5, 7, 12 and 13. In the serum IgE reactivity study, Der p 2, Der p 1 and Blo t 5 had been identified as allergens with both high frequency and magnitude of IgE-binding whereas the other groups were categorized with low IgE-binding frequency and magnitude. Dust samples were obtained by vacuuming 5 areas within each home: bed, carpet, kitchen, sofa, and bedroom floor, using methods described. The range of mean concentration detected for each of the allergen groups (Figure 4) is as follows: Der p 1 (1.5–10.9 μg/g dust) Der p 2 (4.3–7.7 μg/g dust), Blo t 5 (2.5–6.3 μg/g dust), and Der f 13 (1.6–3.5 μg/g dust). Blo t 3 (< 0.2 μg/g dust) and Der p 7 (< 0.6 μg/g dust) concentrations were very low in all sampled 25 areas but still detectable compared to Blo t 12 levels which were below the assay detection limits. Der p 1 and Der p 2 were the dominant allergens found in bed samples. For carpet samples, Der p 1 concentrations were the highest as were Der p 2 in bedroom floor samples. In kitchen, sofa and bedroom floor dust, the allergens found in high concentrations were Blo t 5, Der p 1 and Der p 2, respectively. 12 Allergen concentration (μg/g) 10 Der p 1 8 Der p 2 Blo t 3 6 Blo t 5 Der p 7 4 Der f 13 2 0 0 1 Bed 2 Carpet 3 Kitchen 4 Sofa 5 Floor 6 Dust sample source Dust sample source Figure 4 Distribution of dust mite allergens in Singaporean homes. Data presented as mean concentrations for each allergen (A-F) in each area sampled: beds (n = 51; 53; 26; 36; 26; 36), carpets (n=9; 9; 3; 6; 3; 6), kitchens (n = 7; 9; 5; 15 ;5 ;15), sofas (n = 16; 14; 22; 36; 22; 36) and floors (n = 15; 12; 8; 23; 19; 24). Data for Blo t 12 not shown as readings were below detection limit. 26 For Der p 1, Der p 2, and Blo t 5 allergens, the highest mean concentrations detected ranged from 6.3–10.9 μg/g dust. These amounts of exposure already exceed the reported levels that can be considered as risk factors for sensitization to mites, asthma development and bronchial hyperreactivity in genetically predisposed individuals (Lau et al., 1989; Sporik et al., 1990; Arruda et al., 1991; Fernandez-Caldas et al., 1999). It is also known that the risk of sensitization increases with increasing doses. The mean for Der p 1 found in this study was also higher than previously reported (Zhang et al., 1997). Der p 1 was detected mostly in bed and carpet samples, Der p 2 in bed and bedroom floor samples, Blo t 3 and Der p 7 in sofa samples, Blo t 5 in kitchen and bedroom floor samples while Der f 13 concentrations were equally distributed over all the areas sampled except for sofa samples (Figure 5). The highest concentration recorded in a single sample was for 65 μg/g dust for Der p 1 and 22 μg/g dust for Der p 2. Blo t 5 and Der f 13 both recorded the highest concentration in a single sample of 10 μg/g dust. The most Der p 7 detected in a single sample was 1.8 μg/g dust while Blo t 3 concentration was almost negligible at 0.4 μg/g dust. 27 B Der p 1 70 60 50 40 30 20 10 0 Bed Carpet Kitchen Sofa Floor Allergen concentration (μg/g) Allergen concentration (μg/g) A 22.5 20.0 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0 Der p 2 Bed Dust sample source 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 D Blo t 3 Bed Carpet Kitchen Sofa Floor 10.0 7.5 5.0 2.5 0.0 Bed 1.5 1.0 0.5 Carpet Kitchen Sofa Dust sample source Floor Allergen concentration (μ g/g) Allergen concentration (μg/g) F Der p 7 Bed Carpet Kitchen Sofa Floor Dust sample source 2.0 0.0 Floor Blo t 5 Dust sample source E Sofa Dust sample source Allergen concentration (μg/g) Allergen concentration μg/g) C Carpet Kitchen Der f 13 10.0 7.5 5.0 2.5 0.0 Bed Carpet Kitchen Sofa Floor Dust sample source Figure 5 Concentration of dust mite allergens in dust samples from Singaporean homes. Dust samples collected were assayed for concentrations of the allergens in beds (n=51; 53; 26; 36; 26; 36), carpets (n=9; 9; 3; 6; 3; 6), kitchens (n=7; 9; 5; 15; 5 ;15), sofas (n=16; 14; 22; 36; 22; 36) and floors (n=15; 12; 8; 23; 19; 24). Mean concentration for each allergen is denoted by the horizontal bars in each scatter dot plot. Data for Blo t 12 not shown as readings were below detection limit. 28 Therefore, Der p 1 and Der p 2 can be categorized as having high environmental distribution, Blo t 5 and Der f 13 as moderate and Der p 7, Blo t 3 and Blo t 12 as poorly distributed. The dust data for Der p 1 and Der p 2 support the degree of sensitization shown for both allergen groups in the serum reactivity profile of the Singaporean atopic population. Subsequently, how these allergens interact in a host system was investigated using responder animals in the context of modeling atopic asthma. 3.3 Murine model of dust mite allergen-induced atopic asthma Female 8-week old SPF BALB/c mice representing a responder population were exposed to native Der p 1 and recombinant Der p 2, Blo t 3, Blo t 5, Der p 7, Blo t 12 and Der f 13 allergen proteins according to the exposure protocol described. Native Der p 1 (INDOOR Biotechnologies; UK) was used as the recombinant allergen did not fold properly to preserve its proteolytic activity. For each allergen group studied, 4 immunization doses were tested: 0.5, 1, 2, and 4 μg (except for Blo t 5 where the doses were increased to 1, 5, 10, and 20 μg respectively; refer to explanation is given in section 3.3.1). For every dose, a total of 4 animals were immunized and challenged with the native/recombinant allergen protein. 29 3.3.1 Airway hyperresponsiveness (AHR) Specific response such as airway narrowing to exposure of substances with allergenic properties (e.g. allergens) constitue one of the hallmarks of atopic asthma. Airway hyperresponsiveness is a parameter indicative of the allergen’s capacity to induce lung bronchoconstriction. Assessment of the airway response was performed by challenging the lungs with methacholine, a non-specific agonist. Both asthmatic and non-asthmatic subjects can respond to a non-specific stimulus such as methacholine, but differ in magnitude depending on dose concentrations and airway sensitivity. The animals’ mechanical parameter of airway resistance to methacholine is measured as Enhanced Pause (Penh), a dimensionless unit. In this report, absolute Penh values were converted into “percentage above baseline Penh” indices. The percentage increase above baseline Penh index charts the host’s response towards inhaled methacholine along a dose-response curve calculated based upon the baseline Penh value (whereby baseline Penh is the measurement of pulmonary response to administration of aerosolized PBS solution instead of methacholine). 30 Mice exposed to native Der p 1 protein did not exhibit any specific trending of AHR with increasing methacholine concentrations (Figure 6). At the highest methacholine concentration of 0.1 M, all immunization doses recorded 200–370% increase above baseline Penh except for mice given immunization dose of 2 μg which dipped instead of increasing. Resulting Penh variance between the animals increased with increasing methacholine concentrations. The greatest reactivity was shown for mice given the immunization doses of 1 μg (R2 = 0.9961). Animals given different immunization doses did not display differential AHR towards native Der p 1. This suggests that perhaps a lower immunization dose of native Der p 1 (lower than 1 μg) along with booster immunizations/challenges over a longer period of exposure might elicit a more significant magnitude of response. This would be consistent with published results that only 1 μg of Der p 1 is sufficient to generate an asthma model (Clarke et al., 1999). Der p 1 % increase above baseline Penh 500 400 300 0.5ug 1.0ug 200 2.0ug 4.0ug 100 0 0.01 0.1 1 -100 Methacholine (M) Figure 6 Der p 1-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. 31 Mice exposed to recombinant Der p 2 protein exhibited dose-dependent increased airway hyperresponsiveness with increasing methacholine concentrations (Figure 7). At the highest methacholine concentration of 0.1 M, mice given immunization dose of 4 μg recorded almost 500 % increase above baseline Penh compared to those given the lowest immunization dose of 0.5 μg (almost 300 % increase above baseline Penh). Resulting Penh variance between the animals given the highest immunization dose was also the greatest in comparison to the animals given lower doses. The greatest reactivity were shown for mice given the immunization doses of 2 μg (R2 = 0.9776) and 4 μg (R2 = 0.9797). Der p 2 % increase anove baseline Penh 700 600 500 0.5ug 400 1.0ug 2.0ug 300 4.0ug 200 100 0 0.01 0.1 1 Methacholine (M) Figure 7 Der p 2-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. 32 Mice exposed to recombinant Blo t 3 protein exhibited increased airway hyperresponsiveness with increasing concentrations of methacholine (Figure 8). At the highest methacholine concentration of 0.1 M, mice given immunization dose of 4 μg recorded 250% increase above baseline Penh compared to those given the lowest immunization dose of 0.5 μg (almost 75% increase above baseline Penh). Resulting Penh variance between the animals given the highest immunization dose was also the greatest in comparison to the animals given lower doses. The greatest reactivity was shown for mice given the immunization dose of 4 μg (R2 = 0.9911). Blo t 3 % increase above baseline Penh 300 250 200 0.5ug 1.0ug 150 2.0ug 4.0ug 100 50 0 0.01 0.1 1 Methacholine (M) Figure 8 Blo t 3-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. 33 The initial experiments with Blo t 5 used immunization doses of 0.5, 1, 2, and 4 μg, which failed to produce any clear AHR trending as well as differential response between the doses (data not shown). The immunization doses were thus increased 5-fold to 1, 5, 10, and 20 μg, resulting in increased airway hyperresponsiveness with increasing methacholine concentrations (Figure 9). At the highest methacholine concentration of 0.1 M, mice immunized with doses of 10 and 20 μg recorded almost 325 % increase above baseline Penh compared to those given the lowest immunization dose of 1 μg (almost 30 % increase above baseline Penh). Resulting Penh variance between the animals given the highest immunization dose was also the greatest compared to animals given lower doses. The greatest reactivity was shown for mice given the immunization dose of 20 μg (R2 = 0.8873). Blo t 5 % increase above baseline Penh 500 400 300 1ug 5ug 200 10ug 20ug 100 0 0.01 0.1 1 -100 Methacholine (M) Figure 9 Blo t 5-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. 34 Mice exposed to recombinant Der p 7 protein exhibited increased airway hyperresponsiveness with increasing methacholine concentrations (Figure 10) but with no dose-dependency. At the highest methacholine concentration of 0.1 M, mice given immunization doses of 2 and 4 μg recorded almost 250 % increases above baseline Penh compared to those given the lowest immunization dose of 0.5 μg (almost 50 % increase above baseline Penh). Resulting Penh variance between the animals given the highest immunization dose was also the greatest in comparison to the animals given lower doses. The greatest reactivity was shown for mice given the immunization dose of 4 μg (R2 = 0.9328) while the reactivity graphs of mice administered lower doses appeared to plateau instead or increasing with higher methacholine concentrations. Der p 7 % increase above baseline Penh 350 300 250 200 0.5ug 150 1.0ug 100 2.0ug 4.0ug 50 0 0.01 0.1 -50 1 -100 Methacholine Figure 10 Der p 7-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. 35 Mice exposed to recombinant Blo t 12 protein exhibited increased hyperresponsiveness with increasing concentrations of methacholine (Figure 11), but with higher immunization doses resulting in airway suppression compared to lower immunization doses. At the highest methacholine concentration of 0.1 M, all immunization doses recorded percentage increases above baseline Penh of 300–325%. Resulting Penh variance between the animals given the lowest immunization dose was also the greatest in comparison to the animals given higher doses. The greatest reactivity was shown for mice given the immunization doses of 0.5 μg (R2 = 0.9497). Blo t 12 % increase above baseline Penh 400 350 300 0.5ug 250 1.0ug 200 2.0ug 150 4.0ug 100 50 0 0.01 0.1 1 Methacholine (M) Figure 11 Blo t 12-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. This suppression of AHR with higher immunization dose could be attributed to high-dose tolerance, suggesting that reactivity can be measured with a lower dose range. However, compared to the other allergen groups studied, the 36 Blo t 12 allergen group is not known to be a potent allergen. Thus, it is possible that the intrinsic properties of Blo t 12 allergen work differently in eliciting an immunological response when compared to other allergens studied thus far. Mice exposed to recombinant Der f 13 protein exhibited increased hyperresponsiveness with increasing concentrations of methacholine (Figure 12) but with higher immunization dose resulting in AHR suppression compared to lower immunization doses, similar to the AHR profile of Blo t 12. At the highest methacholine concentration of 0.1 M, mice given immunization dose of 0.5 μg recorded almost 400% increase above baseline Penh. Resulting Penh variance between the animals given the lowest immunization dose was also the greatest in comparison to the animals given higher doses. The greatest reactivity was shown for mice given the immunization dose of 0.5 μg (R2 = 0.9898). Der f 13 % increase above baseline Penh 500 400 300 0.5ug 1.0ug 200 2.0ug 4.0ug 100 0 0.01 0.1 1 -100 Methacholine (M) Figure 12 Der f 13-induced murine AHR. Each dose response curve is plotted with mean data of 4 animals and variance between the 4 animals for each dose is shown as SD error bars on each respective dose response curve. Logarithmic trendlines not shown. 37 When compared with Blo t 12, Der f 13 also appeared to induce AHR suppression at higher immunization doses but with more allergenic strength. Similarly, this suppression of AHR with higher immunization dose could be attributed to the intrinsic properties of the Der f 13 allergen in inducing a different immunological response from the murine host. The Der f 13 allergen is not known to be a potent allergen compared to Der p 1, Der p 2 or Blo t 5. 3.3.2 Sera antibody profile response Besides studying the murine AHR profiles induced by the allergens, serum antibody profiles were investigated to shed light on the allergenicity of the recombinant allergen proteins in inducing a systemic response. Elevated antigenspecific serum IgG1 and IgE titers are hallmark parameters in murine models of atopic asthma (Jungsuwadee et al., 2002; Holt et al., 1991). Mast cells are activated by the cross-linking of allergen-specific IgE on its surface which causes release of inflammatory mediator that contribute towards AHR (Djukanovic et al., 1990) in asthma pathogenesis. IgG1 has also been increasingly implicated in mediating AHR by its capacity to sensitize murine mast cells independent of IgE (Macedo-Soares et al., 2004; Mehlhop et al., 1997; Oshiba et al., 1996), in addition to being the only immunoglobulin class other than IgE in mice that are able to cross-link mast cell FcεRI receptors. Blood samples were collected from each animal before and after the allergen exposure period to obtain pre- and post-exposure sera antibody profiles which were assayed by ELISA using laboratory-raised rabbit antibodies specific against the allergens and commercially available anti-IgG1 and anti-IgE 38 antibodies. Direct ELISA was used to assay for allergen-specific IgG1 antibodies whereas sandwich ELISA was used to detect the scarcer amounts of allergenspecific IgE antibodies. Due to the low levels of OD readings obtained, results in this report were expressed as the difference between OD readings of pre- and post-exposure sera to reflect the change in antibody response. Pre-exposure sera for animals of each allergen exposure were taken as controls against the postexposure sera. For murine IgG1 antibody response (Figure 13), Blo t 3 induced the highest amount of antibodies produced for all immunization doses whereas Blo t 5 induced the lowest amount despite immunization doses being 5 times higher than for all other allergens. Almost all allergens induced production of allergenspecific antibodies in an immunization dose-dependent manner except for Der p 7, which exhibited suppression of IgG1 levels produced at higher immunization doses. For murine IgE antibody response (Figure 14), Der p 2 induced the highest amount of antibodies produced for all immunization doses whereas Der f 13 induced the lowest amount. All the allergens induced production of allergenspecific antibodies in an immunization dose-dependent manner. 39 3.5 3 Difference in OD 2.5 0.5ug 2 1.0ug 2.0ug 1.5 4.0ug 1 0.5 0 Dp1 Dp2 Bt3 Bt5 Dp7 Bt12 Df13 Allergen Figure 13 Allergen-induced murine sera IgG1 profile. Bar graph values plotted as difference between pre- and post-exposure sera OD readings. Sera samples were pooled from 4 animals within each allergen group and dose. For Blo t 5, immunization doses were 1, 5, 10, and 20 μg respectively. 0.9 0.8 Difference of OD values 0.7 0.6 0.5ug 0.5 1.0ug 0.4 2.0ug 4.0ug 0.3 0.2 0.1 0 Dp1 Dp2 Bt3 Bt5 Dp7 Bt12 Df13 Allergen Figure 14 Allergen-induced murine sera IgE profile response. Bar graph values plotted as difference between pre- and post-exposure sera OD readings. Sera samples were pooled from 4 animals within each allergen group and dose. For Blo t 5, immunization doses were 1, 5, 10, and 20 μg respectively. 40 Native Der p 1, and Der p 2 induced about twice as much IgG1 compared to IgE in allergen-exposed mice. Blo t 3 induced 5 times more IgG1 than allergenspecific IgE. Blo t 5 on the other hand produced approximately the same amount of both allergen-specific antibodies even with immunization doses 5 times more than the other mice exposed to the rest of the allergens. Der p 7 produced about 6 to10 times more IgG1 than IgE at lower doses but approximately the same amount of both antibodies at higher doses. In Blo t 12- and Der f 13-exposed mice, production of IgG1 was about 10 times more than IgE levels. Consequently, the predominant production of IgG1 rather than IgE with these two groups may characterize the AHR response seen with the two allergen groups. Despite having lower allergenic potency, Blo t 12 and Der f 13 allergens are able to induce AHR at lower doses via IgG1 rather than IgE. However, further characterization is needed to provide more conclusive explanation regarding the suppression of airway responses with higher doses of Blo t 12 and Der f 13. 3.3.3 Lung histology studies Distended murine lungs kept in 10% formalin solution were processed and tissues embedded in paraffin wax. Sections were cut with a microtome to about 5 7 microns thick, dewaxed and mounted on microscopic slides before being stained with hematoxylin and eosin dyes. Hematoxylin visualizes the nucleic acids of cell nuclei and eosin stains the cell cytoplasmic components. Slides were observed under the microscope for changes indicative of allergen-induced inflammation in 41 the lungs: cellular infiltration such as macrophages and eosinophils, mucus hypersecretion or smooth muscle hypertrophy. However, microscopic slides of lung sections of all allergen-exposed mice studied did not reveal any significant inflammation observations when compared to lung sections from control mice exposed to phosphate buffered saline (PBS). Mice are known to develop AHR without histologic airway inflammation when the allergen is administered only through the respiratory route (Hessel et al., 1995; Renz et al., 1992). In this study, animals were first immunized via an intraperitoneal injection of allergen solutions without adjuvant, followed by a booster intraperitoneal injection and subsequent 4 daily intranasal challenges. Thus the lack of lung inflammation observed may be attributed to the route of allergen administration employed as well as the low dosage of allergen given in the absence of adjuvants. The short exposure duration and subsequent low levels of serum IgE induced may also be insufficient to affect any significant lung histology changes. 42 Chapter 4: Conclusion In this study, it was initially observed that different dust mite-sensitive individuals in an atopic population reacted to different groups of dust mite allergens with varying degrees of magnitude and frequency of IgE-binding. A panel was created with selected allergens of different species representing the major dust mite allergen groups: Der p 1, Der p 2, Blo t 3, Blo t 5, Der p 7, Blo t 12, and Der f 13. Selection criteria were based on the known IgE reactivity profile of the allergens and characterization information already published or available in the laboratory. The studies of the serum allergen-specific IgE reactivity in the local atopic population and the environmental distribution of dust mite allergens showed interesting patterns for the different allergen groups inducing differential host immunological responses. The specific dust mite allergen-induced responses for each of the allergen groups studied are summarized in the following paragraphs: Major allergens such as groups 1 and 2 had specific environmental niche concentrations with high exposure (distribution concentrated in bed and bedroom floor samples) that directly corresponded to the magnitude and frequency of IgEbinding. Thus, the capacity of major allergen groups 1 and 2 in invoking an immune response are expected to be high. Futhermore, there are studies that suggested that the proteolytic activity of the group 1 allergens may provide an adjuvant effect such as the cleavage of CD23 (Schulz et al., 1997; Hewitt et al., 1995) which is important in the regulation of IgE responses. However, native Der p 1 did not induce a clear AHR in Der p 1-exposed mice. Immunization with 43 Der p 1 produced twice as much IgG1 than IgE. The lack of significant AHR may be attributed to the fact that atopic mice produce different immune responses towards allergenic fractions of a mite extract. High molecular weight-allergenic fraction (HM1) abundant in D. farinae extracts aggravated AHR in mice rather than the HM1-depleted fecal extract (Tategaki et al., 2002). Der p 1 is known to be about 25kDa in size, which is excluded from the range of the high-molecularweight allergenic fraction. Sera IgE reactivity and house dust data corroborated the role of group 2 as a major dust mite allergen group. Der p 2 also induced dose-dependent increased AHR in immunized mice with production of IgG1 twice the amount IgE. These data suggest that high amounts of group 2 allergens in concentrated exposure areas such as beds, and the intrinsic allergenicity of the protein elicit more significant atopic presentations compared to major allergen group 1. Group 13 allergens are most similar in size and function compared to group 2 allergens. Der f 13 is known to be a fatty acid binding protein (FABP) of about 14 kDa in size. High concentrations of group 13 allergens in the environment do not increase its sensitization capacity or its ability to elevate AHR. While group 2 exhibited immunization dose-dependent increase of AHR response to methacholine, group 13 suppressed AHR with increasing immunization doses at comparable reactivity levels. Similar to group 7, group 13 also produced ten times more IgG1 compared to IgE titers in immunized mice. This suggests a role for the immunoglobulin in mediating inflammatory responses. A possible explanation for this suppression observation may attributed to the function and structure similarity of group 13 to the family of lipocalin proteins. Its effective 44 dispersion in environmental dust, low stimulation capacity for T-cell proliferation (unpublished data), endogenous FABPs capacity to bind IgE and positive skin prick test results (Chan et al, 2006) support a possible role as an allergen with immunomodulatory properties. Group 12 allergen also suppressed AHR at higher immunization doses but with less airway reactivity magnitude than group 13 allergens. Group 12 also share size similarity with group 2 and 3 allergens. Among the low IgE-binding capacity dust mite allergens, group 12 showed the highest frequency but the lowest magnitude. The IgG1 levels induced in Blo t 12-exposed mice were 10 times greater than the titers of IgE produced. With its scarce distribution in house dust compared to major groups such as 1 or 2, it can only be inferred that although not very allergenic, high immunization doses of group 12 produces high IgG1 levels that may possibly modulate AHR response in mice. The lack of published work on group 12 allergens thus far and its unknown function do not allow more complete information on its allergenicity. Group 7 allergen demonstrated the ability to induce AHR response with increasing methacholine concentrations and to sensitize an atopic population. It is the only allergen group to demonstrate a reversal in allergen dose-dependent production of IgE and IgG1, with titers of IgG1 being 6–10 times more than IgE titers at lower doses, which warrants further investigation. It can only be inferred that group 7 allergens are immunogenic and can elicit immune responses different from the major allergen groups such as 1 and 2. It is also possible that group 7 induces immunologic tolerance (Platts-Mills et al., 2000) at low sensitization 45 doses. However, the IgE-binding capacity of Group 12 was not significant enough to describe its allergenicity. Group 5 allergens have both high IgE-binding frequency and magnitude but are not as well represented in the environment as well as the group 1 and 2 allergens. However, compared with the relatively low reactivity and distribution of Group 3 and 12 allergens, it certainly has a higher sensitization risk. Nonetheless, substantial environmental concentrations and IgE-binding capacity do not correlate with its ability to invoke a systemic immune response. It required 5 times more allergen than group 3 in order to induce a clear AHR response. At the immunization dose of 10 μg, group 5 induced only slightly more airway reactivity than the magnitude induced by group 3 at the immunization dose of 1 μg. It can be concluded that Group 5 allergens depend on high exposure levels in order to invoke a stronger immune response. Group 3 allergen’s low allergenicity may be attributed to it being highly susceptible to degradation in the environment (Stewart et al., 1989). Size of the allergen may also play a role as group 3 allergens are almost twice as large as Group 5 ones. What we may conclude at this stage is that clearly, the different groups of allergens interact specifically and differently with the host to induce immunological responses. Combined observations from the local population sera IgE reactivity screen, the environmental dust screens and the immunological responses of the murine model of atopic asthma pointed towards the inherent properties of the dust mite allergen groups as the source of these different responses. It is of great interest to study how well-dispersed allergens with low concentrations such as group 5 and 13 (both with opposite sensitization profiles) 46 affect the pathogenesis of atopic diseases. These groups which are less wellcharacterized compared to Groups 1 and 2 may yield insights into the dust mite allergens which are clinically important triggers for bronchial asthma in the context of the typical Singapore household and sensitization patterns. Comparison studies can also be drawn between group 2 and 13 allergens with their structure and function similarity but opposite sensitization and AHR profiles. More characterization work needs to be done to elucidate the functions of these allergens which would help to determine why some people are not allergic and how the allergen-host interaction plays out in the pathogenesis of atopic diseases such as asthma. 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Hum Mol Genet. 8(4):601-5. 58 [...]... sequence data available in WHO/IUIS list of allergens or GenBank c Data for Mag allergen d Data for recombinant Mag 3 allergen e Data for natural Mag 3 allergen f Not listed in WHO/IUIS list of allergens but published and sequence data available in GenBank 6 More than 95 % of the allergen accumulated in mite cultures is found in fecal particles (Tovey et al., 1981), which makes mite feces a major source of. .. is also a lack of animal models using dust mite allergens as the allergen source (Sharma et al., 2003) The available studies of atopic asthma using dust mite allergens have mostly been limited to house dust mite extracts (Tategaki et al., 2002; Tumas et al., 2001) rather than the use of recombinant proteins The content of extracts includes a variety of allergenic and non-allergenic components which are... itself, many advances in understanding the mechanism of asthma and allergy have been made with the use of murine models These studies have also proven useful in characterizing specific allergen- induced immunological responses and immunological properties of allergens BALB/c and C57BL/6j are two of the commonest strains of mice used in studies of allergies and atopic asthma One of the main factors to... 2001) Allergen -specific IgE is measured as risk factor for asthma as well as the allergenicity of the allergen whereas IgG1 is measured because it is also able to bind mast cells and basophils to cause degranulation (Tumas et al., 1991) Experimental protocols mostly differ in the age of animals used, dose and type of allergen, route of allergen administration, length of allergen exposure and method of. .. university Animal Holding Unit Food and water were provided ad libitum Animals were sacrificed by chemical euthanasia after the final harvest and disposed off as biohazard waste according to biosafety guidelines Immunization was administered to the animals subcutaneously using 300 μg of recombinant protein diluted in a mixture of 500 μl of PBS and equal volume of Freund’s complete adjuvant (Sigma-Aldrich;... the dust mite allergen groups have been elucidated except for groups 2, 5, 7, 12 and 21 Their diverse biological functions include enzymes, enzyme inhibitors, ligand binding proteins and structural proteins Dust mite allergens are one of the most important aeroallergens inducing asthma and are much more relevant than ovalbumin which is the standard antigen used in murine models of atopic asthma There... house dust allergen Dust mite allergens have already been detected in household niches worldwide For an atopic individual, it takes lesser amounts of allergens to invoke an immune response compared to a non-atopic individual Studies have previously been conducted and are also ongoing to correlate the amount of allergen found in environmental dust with the risk of allergen sensitization Many functions of. .. Blomia tropicalis, Dermatophagoides pteronyssinus and Dermatophagoides farinae (Chew et al., 1999) Although asthma is a complex multifactorial disease, atopy presents a vital risk factor for asthma, especially with the most significant period of allergy sensitization development to allergens being in early childhood (Peden, 2002) A summary of the mechanism of allergy in the pathogenesis of atopic asthma... Murine models of atopic asthma The Mouse Genome Project has revealed that mice and humans both have about 30,000 genes and share 99% of those genes alike About 1,200 new genes were discovered in the human genome because of mouse-human comparisons (90 % of genes associated with diseases are identical in human and mouse) The availability of well-characterized mutants and inbred strains provide a wealth... study also provided materials (lungs, sera and BAL fluid from mice immunized with and exposed to dust mite allergens) for future functional genomic and proteomic characterization of dust mite allergen- induced responses in a host immune system Such future characterization will yield possible clues into putative molecular markers or pathways of target in the pathogenesis of atopic asthma The main deliverables ... relevant than ovalbumin which is the standard antigen used in murine models of atopic asthma There is also a lack of animal models using dust mite allergens as the allergen source (Sharma et al.,... these allergens interact in a host system was investigated using responder animals in the context of modeling atopic asthma 3.3 Murine model of dust mite allergen- induced atopic asthma Female 8-week... recombinant Mag allergen e Data for natural Mag allergen f Not listed in WHO/IUIS list of allergens but published and sequence data available in GenBank More than 95 % of the allergen accumulated in mite